Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.     

Quantitative cytology of the alfalfa generative cell and its relation to male plastid inheritance patterns in three genotypes

Summary Studies utilizing restriction analysis of plastid DNA, as well as those employing chlorophyll-deficient mutants, have shown a high frequency of paternal plastid transmission in alfalfa. Recent research has also shown that plastid inheritance patterns among alfalfa genotypes and are under genetic control. In a previous study we were unable to detect any correlations between qualitative, three-dimensional ultrastructure of generative cells and male plastid transmission strength in certain genotypes. In the present study we used serial ultrathin sectioning, computerized reconstruction and quantitation, and stereology to further analyze generative cells within mature pollen. Measurements included volumes and surface areas of cells, nuclei, and organelles, as well as organelle number and distribution. Three genotypes were investigated, one that is a strong transmitter of male plastids (genotype 301), one that is a weaker transmitter of male plastids (genotype 7W), and a third that is an even weaker male plastid transmitter (genotype MS-5). Our results show that genotype MS-5 has significantly fewer plastids/generative cell than either of the other genotypes, which may account for it being a relatively poor transmitter of male plastids. However, plastid number does not explain known differences in male plastid inheritance between genotypes 301 and 7W, since plastid number does not differ significantly between these two genotypes. Regarding the other features of generative cells measured in this study, no consistent correlations were found that might account for differences in male plastid inheritance patterns between genotypes. Plastid distribution is equal in each end of the spindle-shaped generative cell in all genotypes studied. Similar relative results were found with regard to mitochondria within generative cells; however, comparative genetic data are not available on mitochondrial transmission patterns in alfalfa genotypes.     

Quantitative ultrastructural analysis of barley sperm

Summary Complete serial ultrathin sections of seven sperm pairs, computer-assisted measurements of cell, nuclear and organelle surface areas and volumes, and three-dimensional imagery were used to demonstrate that a process of cytoplasm and organelle elimination occurs during sperm maturation in barley. The number of mitochondria per sperm cell is reduced by 50%; sperm cell surface area and volume are reduced by 30% and 51% respectively. Mean volume and surface area per mitochondrion are significantly less in mature sperms. No examples of mitochondrial fusion or degeneration were observed within sperm cells. These data, along with observations of plasma membrane apposition and vesiculation within cytoplasmic extensions containing mitochondria, support the proposition that cytoplasm and organelle loss results primarily from the formation of cytoplasmic projections that are subsequently discarded from the sperm cell body. Comparisons of the quantitative data, including the number of mitochondria, indicate that differences between sperm cells of a pair are absent to very slight. Spatial organization within the pollen grain is such that the mature sperms, as well as the sperms and vegetative nucleus, are not in close proximity.     

Receptor dynamics of closely related ligands: "fast'' and "slow'' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.     

Quantitative three-dimensional study on the position of the female gametophyte and its constituent cells as a prerequisite for corn (Zea mays) transformation

Summary The position of the embryo sac in the spikelet and of the embryo sac's constituent cells within the sporophytic tissues of Zea mays was localized by scanning electron microscopy, serial thick sectioning, and computer three-dimensional reconstruction. Within certain limits, the embryo sac is consistently oriented in the same position inside of the spikelet. This information is a prerequisite for successful microinjections into the in situ female cells of Zea mays.     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts

Summary Isolated egg cell protoplasts ofZea mays L., inbred line A 188, have been studied at the transmission electron microscope level. Their preparation for electron microscopy has been performed by embedding in ultra-low gelling agarose as a preliminary step. Five isolated egg cell protoplasts were serially ultrathin sectioned and studied in detail. One of these protoplasts was reconstructed in three dimensions to provide additional information on its structure. After enzymatic digestion and microdissection, isolated egg cells are true, highly vacuolized protoplasts. The structure of their organelles agrees with in situ observations, indicating an ultrastructural intactness after isolation: the mitochondria are polymorphic, form reticulate networks, and have well developed cristae; the plastids contain starch grains; and the spherical nucleus is euchromatic. As in situ, the organelles of the isolated egg cell protoplasts are aggregated near the nucleus. The complete picture provided by this work should serve as a comparative base for studies on in vitro fertilization products.     

ULTRASTRUCTURAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE OVULES OF SAINTPAULIA IONANTHA (GESNERIACEAE) AND ITS RELATION TO SYNERGID FUNCTION AND EMBRYO SAC NUTRITION

Ovules of African violet were analyzed for adenosine triphosphatase activity. Ovules from unpollinated flowers of three different ages were fixed in buffered, 3% paraformaldehyde, incubated in the Wachstein-Meisel medium, and processed for electron microscopy. Results showed a heavy reaction product in the endothelium and inner micropylar cells of the integument with decreasing amounts elsewhere. Reaction product was localized primarily on the plasma membrane, and occasionally in the nuclear membrane, endoplasmic reticulum, small vacuoles, and mitochondria. The synergids, egg, central cell, and antipodals were essentailly devoid of reaction product except for rare occurrences in the smaller vacuoles and mitochondria of the synergids, and fragmentary deposits on the plasma membrane of the antipodals. No differences were found in any of the floral stages examined. These results suggest that the integumentary cells nearest the embryo sac are equipped with the necessary enzymes for active translocation of solutes into the embryo sac and that the cells of the megagametophyte apparently function more passively in this regard.     

Enhanced expression of cellular receptors for human interferon α on peripheral lymphocytes from patients with down''s syndrome

The sequence of 370 bases at the 5′-end of bovine thyroglobulin mRNA has been determine. A41 base untranslated segment was found preceeding the ATG initiator codon. It is followed by an open reading frame providing the first data on thyroglobulin primary structure. Analysis of the amino acid sequence demonstrated the presence of an 18 residue hydrophobic segment representing a putative signal peptide. Comparison of the amino terminal sequence of thyroglobulin with that of peptides known to contain thyroid hormones [7,8] demonstrated that the first tyrosine in native thyroglobulin is mainly found as thyroxine in the mature iodinated protein [8]. Our results clearly identify the amino-terminal region of thyroglobulin as an important hormonogenic domain of the protein.